Quantitative analysis of triglyceride species of vegetable oils by high performance liquid chromatography via a flame ionization detector.

A method for the quantitative analysis of triglyceride species composition of vegetable oils by reversed-phase high performance liquid chromatography (RP-HPLC) via a flame ionization detector (FID) is described. Triglycerides are separated into molecular species via Zorbax chemically bonded octadecylsilane (ODS) columns using gradient elution with methylene chloride in acetonitrile. Identification of species is made by matching the retention times of the peaks in the chromatogram with the order of elution of all of the species that could be present in the sample on the basis of a random distribution of the fatty acids and comparison of experimental and calculated theoretical carbon numbers (TCN). Quantitative analysis is based on a direct proportionality of peak areas. Differences in the response of individual species were small and did not dictate the use of response factors. The method is applied to cocoa butter before and after randomization, soybean oil and pure olive oil.

Energetic efficiency and mitochondrial function in rats fed trans fatty acids.

Nutritional experiments with growing rats were conducted to study the effect of dietary t18:1 and t,t18:2 in mixtures with safflower oil (77.1% linoleate) on oxidative phosphorylation of isolated liver mitochondria and on energetic efficiency as measured in energy balance experiments. Partially hydrogenated soybean oil (PHSO) with 45% t18:1 and an ethyl ester concentrate of trans fatty acids (TRANS) containing 52% t,t18:2 were used. Six groups of male rats were fed diets with 10% (wt/wt) fat supplements: 1, safflower oil (SAF); 2, 8% PHSO + 2% SAF; 3, 0.5% TRANS + 9.5% SAF; 4, 1% TRANS + 9% SAF; 5, 2% TRANS + 8% SAF; and 6, 5% TRANS + 5% SAF. These fat supplements contained 77.1, 17.1, 73.3, 69.4, 61.7 and 38% linoleate for groups 1-6, respectively. The basal metabolic rate was not affected by dietary treatments, neither were the metabolizable energy values. As compared to the SAF group, rats receiving the 8% PHSO + 2% SAF showed no different effect on energy utilization or mitochondrial respiratory function. TRANS was found to reduce the efficiency of metabolizable energy utilization at the 5% level (group 6), while mitochondrial ATP synthesis was significantly depressed in both the 2% and the 5% TRANS groups. A parallelism was observed between t,t18:2 incorporation in liver mitochondria, reduced mitochondrial oxidative function and depressed energetic efficiency. The experiments indicated that t,t18:2 exerted a significantly negative effect on energy utilization at 1.0 and 2.5% levels in the diet even when fed with relatively high concentrations of linoleate.

Analysis of triglyceride species by high-performance liquid chromatography via a flame lonization detector.

The analysis of triglyceride species by high performance liquid chromatography (HPLC) with a flame ionization detector (FID) and reversed-phase chromatography using chemically bonded octadecyl silane (ODS) Zorbax columns and gradient or isocratic solvent elution with methylene chloride/acetonitrile is described. Triglycerides containing acyl groups of critical pairs,trans and positional isomers, as well as mixtures of even and odd chain lengths are separated. Identification of triglycerides is made on the basis of retention times compared with equivalent and theoretical carbon numbers, and comparison with chromatograms of reference triglyceride mixtures. The methodology is demonstrated by fractionizing the triglycerides of olive oil under different chromatographic conditions using single and coupled conventional 250×4.6 mm columns and a short 80×6.2 mm column for fast separations.

Hepatic fatty acids and acyl desaturases in rats: effects of dietary carbohydrate and essential fatty acids.

For a 9-week period, six groups of weanling male rats were fed either a fat-free diet or a diet containing 10% hydrogenated coconut oil (HCO) or 10% safflower oil (SAF), respectively. Each of these diets contained either glucose or sucrose as the only carbohydrate. In the fat-free diets, the carbohydrate level was 67.3% and in the fat-supplemented diets 44.8%. Including HCO in the fat-free diet did not significantly alter hepatic 5-, 6-, and 9-desaturase activity, whereas addition of SAF significantly depressed all these activities. As compared to glucose, sucrose induced higher 9-desaturase activity in the rats on the fat-free diets. Adding HCO or SAF to the diet, simultaneously with lowering the carbohydrate level, diminished the stimulatory effect of dietary sucrose versus glucose on 9-desaturase activity. Levels of 20:4n6 and 20:3n9 in the fatty acid profiles of the liver microsomes were not influenced by dietary carbohydrate source, neither were the activities of the 5- and 6-desaturases, providing indirect evidence that dietary sucrose, as compared to glucose, did not differently affect biosynthesis of 20:4n6 and 20:3n9.

Effects of dietary long-chain fatty acids on the rat biosynthesis of unsaturated fatty acids in the rat.

Menhaden oil (ME) was included in semisynthetic diets to study the effect of long-chain fatty acids, mainly 20:5n3 and 22:6n3, on the biosynthesis of polyunsaturated fatty acids and on the 6- and 9-desaturase activities in liver microsomes. Five experimental diets, with the following fat supplements, were fed to male rats from weaning for a period of 33 weeks: 5% safflower oil (SAF) + 10% hydrogenated coconut oil (HCO), 5% SAF + 5% HCO + 5% ME, 5% SAF + 10% ME, 15% HCO and 5% HCO + 10% ME. The last two diets were deficient in linoleic acid. The three nondeficient diets contained similar amounts of linoleic acid. Including ME in the diets depressed the 6- and 9-desaturase activities, especially in the linoleic acid--deficient rats. The synthesis of 20:4n6, 22:4n6 and 22:5n6 were depressed. These effects were related to the preferential accumulation of dietary 20:5n3 and 22:6n3 in the liver microsomes, as compared to the n6 fatty acids. It is hypothesized that dietary 20:5n3 and 22:6n3 increase the minimum requirement for linoleic acid in the diet.

Interrelationship between dietary trans Fatty acids and the 6- and 9-desaturases in the rat.

Studies are reported on the effects of dietary trans fatty acids on the 6- and 9-acyl desaturase activities in the liver microsomes of rats fed essential fatty acid (EFA)-deficient and non-EFA-deficient diets. In experiment I, weanling male rats were fed a semisynthetic diet with either 10% safflower oil (SAF) or 10% hydrogenated coconut oil (HCO). At the age of one year, half of the dietary fat was replaced by a supplement containing elaidate, linolelaidate and cis, trans-trans,cis-18:2 (TRANS) for 12 weeks. In experiment II, male rats which were kept from weaning on a 10% (SAF + TRANS, or 5% HCO + 5% TRANS. Feeding TRANS depressed the 6-desaturase activity in the liver microsomes, especially in the EFA-deficient rats (HCO + TRANS group of experiment I). Unlike the 6-deaturase activity, the 9-desaturase activity was not inhibited by the dietary trans fatty acids and was significantly stimulated in the non-EFA-deficient rats (SAF + TRANS group of experiment I and HCO + TRANS groups of experiment II). This was evidence by incubation reaction and by comparisons of fatty acid consumptions and microsomal fatty acid levels, showing extra biosynthesis of 16:1 and 18:1 when TRANS was fed. The biosynthesis of essential (n-6) fatty acids was depressed by the TRANS supplement in EFA-deficient as well as in non-EFA-deficient animals.

Covalent binding of peroxidized linoleic acid to protein and amino acids as models for lipofuscin formation.

The fluorescent substances produced by the reaction of linoleic acid hydroperoxides (LOOH) with ca. 20 different amino acids and bovine serum albumin (BSA) were studied. Only the amino acids, lysine, glycine, arginine, histidine and phenylalanine, gave products with strong fluorescent properties. Products of lysine had a fluorescence intensity of ca. 10 times those of glycine and 100 times those of phenylalanine. The N-acylation of amino acids greatly reduced the fluorescence of the products of the reaction except lysine and arginine. The fluorescence of the products of the reaction of LOOH with N-acetyl BSA was only ca. 25% of the control BSA under the same conditions. It appeared that the substances formed from the reaction of LOOH with BSA were crosslinked polymers as evidenced by column chromatography and polyacrylamide gel electrophoresis. These products were insoluble in common organic solvents and their fluorescent intensities correlated well with the thiobarbituric acid (TBA) test. These observations appear to be highly important in the formation of lipofuscin substances, particularly those associated with the aging pigments which accumulate during aging in mammalian tissues.

Quantitative analysis of lipid classes by liquid chromatography via a flame ionization detector.

A method is described for the direct quantitative analysis of the lipid classes of mammalian tissue lipids using high performance liquid chromatography (HPLC) with a flame ionization detector (FID). The lipid is extracted from the tissue with chloroform/methanol after deactivation of hydrolytic enzymes and removal of nonlipid substances by extraction with hot dilute acetic acid (0.05N). Separation of the lipid classes is performed with a column (45 cm × 0.2 cm id) of 8 µm silica (Spherisorb, Phase Sep, Hauppague, NY) treated with concentrated ammonium hydroxide at a solvent flow rate of 0.5 ml/min, which requires a pressure of ca. 900 psi. Cholesteryl esters (CE) and triglycerides (TG) are eluted first with Skellysolve B/methylene chloride (1∶1, v/v); cholesterol (CH) is eluted with chloroform/methylene chloride (1∶2, v/v) and the phospholipids with methanol containing 6% ammonium hydroxide added to the latter solvent in a linear gradient. The neutral lipids are eluted in ca. 12 min and the phospholipids in an additional 30 min. The relative amount of each lipid class was determined from standard curves of the peak areas obtained according to response factors using erucyl alcohol as an internal standard. The method was applied to samples of kidney, liver and serum of rats. Duplicate analyses were generally within ca. 1.0% and good agreement was obtained in the analysis of the lipid classes of Azolectin and liver mitochondria lipid compared to thin layer chromatography (TLC) via photodensitometry of charred spots or phosphorus analysis of recovered phospholipids.

Effects of dietary trans acids on the biosynthesis of arachidonic acid in rat liver microsomes.

Effects of dietary trans acids on the interconversion of linoleic acid was studied using the liver microsomal fraction of rats fed a semipurified diet containing fat supplements of safflower oil (SAFF), hydrogenated coconut oil (HCO) at 5 and 20 at and 20% levels or a 5% level of a supplement containing 50.3% linolelaidic and 24.3% elaidic acids devoid of cis,cis-linoleic acid (TRANS). Growth rate was suppressed to greater extent with the animals fed the 20% than the 5% level of the HCO-supplemented diets and still further by the TRANS diet compared to the groups fed the SAFF diets. Food intake was greater in the groups fed the HCO than the SAFF-supplemented diets, demonstrating the marked effect of an essential fatty acid (EFA) deficiency on feed efficiency. In contrast to an EFA deficiency produced by the HCO supplement, which stimulated the in vitro liver microsomal biosynthesis of arachidonic acid, diets containing the TRANS supplement exacerabated the EFA deficiency and depressed 6-desaturase activity of the liver microsomal fraction. The liver microsomal fraction of the animals receiving this supplement also was more sensitive to fatty acid inhibition of the desaturation of linoleic acid than those obtained from animals fed either the SAFF or HCO diets. It is suggested that dietary trans acids alter the physical properties of the 6-desaturase enzyme system, suppressing its activity, which increases the saturation of the tissue lipids and, in turn, the requirement for EFA or polyunsaturated fatty acids.

Histopathologic study of adverse effects of linoleic acid on rat lung.

Linoleic acid administered intravenously was highly toxic to the rat lung. The severity of the toxicity paralleled the dosage administered and was characterized by generalized, irreversible damage to alveolar wall structures. Although evidence of tissue damage was not apparent or minimal by light microscopy, ultrastructural studies disclosed extensive degeneration and necrosis of septal wall components with 15 minutes following injection of low dosages of linoleic acid. Degeneration of endothelial cells was most severe suggesting that these cells were the target of injury resulting in impaired vascular integrity, interstitial and alveolar edema, and degeneration of type 1 pneumocytes. Fragmentation and stripping of endothelium from capillary basement membranes was uniformly present and was in some instances associated with platelet aggregation indicative of early thrombosis. These studies suggest that this toxic effect on septal capillaries leading to increased permeability and alveolar edema may be a contributing factor to death occurring from free fatty acid toxicity in the human.

Isolation and analysis of age-related fluorescent substances in rat testes.

Fluorescent substances were extracted from rat testicular tissue with 2,2-dimethoxypropane (DMP) and analyzed by 2-dimensional thin layer chromatography (TLC). One substance that accumulated with increasing age of the animals was isolated and analyzed quantitatively by spectrophotofluorometry using quinine sulfate as a standard. This substance, which was designed as an age-related fluorescent substance (ARFS), exhibited an excitation maximum at 355 nm and an emission maximum at 490 nm. Its fluorescence was quenched by metal chelators and at alkaline pH, indicating it contained a conjugated Schiff base structure. Quantitative analysis of this substance in the testes of rats 1, 2, 11 and 20 months of age showed that it increased linearly with age. The relation of this substance to aging also was indicated by its detection in animals of different ages fed diets of both low and high unsaturation.

Inflammation in acne vulgaris: leukocyte attraction and cytotoxicity by comedonal material.

The chemoattraction of comedonal material for leukocytes was evaluated. Material from open comedones attracted mononculear leukocytes but did not attract polymorphonuclear leukocytes. At higher concentrations, comedonal material was cytotoxic for leukocytes of both types. Of the comedonal components tested, free fatty acids produced the greatest cytotoxicity. The attraction and killing of leukocytes by comedonal components may be the mechanisms for the initiation or the enhancement (or both) of inflammation in acne vulgaris.

A simplified procedure for the quantitative extraction of lipids from brain tissue.

A method is described for the quantitative extraction of lipid from brain tissue with chloroform/methanol (C/M) that eliminates secondary purification of the lipid extract by dextran-gel chromatography or aqueous washing of the organic extract. Nonlipid substances that generally contaminate C/M lipid extracts are separated by pre-extraction of the tissue with dilute (0.25%) aqueous acetic acid. The residual tissue is extracted twice with 40 volumes of C/M (1:1, v/v). Approximately 97% of the lipid is recovered in these extractions. A third extraction which yields ca. 1% more lipid is performed if the process is discontinued at this stage in a shortened version of the method. The remainder of the lipid is recovered after treatment of the tissue with 1 N HC1 by two additional extractions, the first with 40 volumes of C/M (1:2, v/v) and the second with 40 volumes of methanol. The method, which was demonstrated with pig brain, gave a complete extraction of the lipid, including gangliosides, free of nonlipid substances.

Effects of essential fatty acid deficiency on prostaglandin synthesis and fatty acid composition in rat renal medulla.

Studies are reported on the capacity of isolated rat renal papilla (inner medulla) to synthesize and release prostaglandin (PG) E from endogenous and exogenous precursor(s) during development of an essential fatty acid (EFA) deficiency in the rat. Weanling (21-day-old) male Sprague-Dawley rats were fed a fat-free diet supplemented with either 5% hydrogenated coconut oil (HCO) or 5% safflower oil (SO). At approximately 3, 6 and 7 weeks (6. 9 and 10 weeks of age), groups of animals fed each diet were killed for studies of PGE synthesis in the renal papillae. Differences in the fatty acid composition of the papillae lipids of the animals of each group were also determined. The in vitro production of PGE from endogenous precursor(s) was significantly reduced in the papillae from the 6-week-old rats fed the HCO diet compared to the control (SO) rats, and appeared to be near maximally depressed in the 10-week-old animals compared to that of animals fed an EFA deficient diet for over a year in an accessory experiment. Analyses of the fatty acids of the papillae lipids of the HCO groups showed that the levels of 18:2 and 20:4 were markedly reduced, and those of 16:1, 18:1 and 20:3 were elevated compared to the controls even in the 6-week-old animals, typical of an EFA deficiency. The papillae lipids of the animals fed the HCO diet were also depleted of their stores of 22:4 omega 6. A fatty acid believed to be derived by chain elongation of 20:3 omega 9, 22:3, was found in large concentrations in the papillae triglycerides of the EFA deficient rats. Incubations of exogenous arachidonic acid (20:4) in homogenates and tissue slices of the papillae of the HCO dietary groups showed that the PG synthetase was not impaired by an EFA deficiency. The rate of PGE synthesis in the papillae of the EFA deficient animals was generally enhanced when exogenous 20:4 was added, indicating that the concentration of available precursor(s) is a primary factor in the control of PGE synthesis in the papilla of the rat.

Effect of hypophysectomy of cholesteryl ester synthesis and hydrolysis in testes and on serum lecithin-cholesterol acyltransferase activity in rats.

Studies are reported of the effect of hypophysectomy on cholesterol esterase activity of testicular tissue and serum lecithin-cholesterol acyltransferase (LCAT) activity in rats. The testes of male Sprague-Dawley rats of 200-255 g were excised from animals sacrificed at 3, 7, and 15 days after hypophysectomy. Assays for cholesterol-esterifying and hydrolytic activities of the testicular tissues of these animals, compared to control animals, showed that hypophysectomy decreased both cholesteryl ester synthesis and hydrolysis. Hydrolytic activity was affected to a greater extent than esterifying activity. LCAT activity was significantly decreased by hypophysectomy compared to that of control animals. Although serum LCAT and testicular cholesterol esterase activities were decreased, the overall effect of hypophysectomy produced an increase in the level of serum cholesterol and cholesteryl esters. It is suggested that the role of essential fatty acids (EFA) in testicular function is related to the utilization of cholesteryl esters in androgen synthesis.